Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signaling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signaling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analyzed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared to IFM (1.330.19, ratio of SNO content Perf-SSM vs Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.720.17 and 1.070.04, ratio of SNO content IPC-SSM vs Perf-IFM and IPC-IFM vs Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3 and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3-/- mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM are the major target for protein SNO in the IPC mouse heart, suggesting that the SSM may be the preferential target of sarcolemmal signaling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in cardioprotection. We have also begun to explore the role of sulfhydration in cardioprotection. hydrogen sulfide (H2S) and nitric oxide (NO), play important roles in postconditioning (PostC)-induced cardioprotection. The emerging data suggest that both H2S and NO could regulate protein function through redox-based protein post-translational modification on cysteine residue(s), i.e., S-sulfhydration (SSH) and S-nitrosylation (SNO), respectively. In this study, we examined whether there is a synergistic protective effect in pharmacological PostC mouse hearts with H2S and NO donors using Langendorff perfused heart model. After 20 min of equilibrium perfusion and 20 min of no-flow global ischemia, the heart was subjected to pharmacological PostC at the beginning of reperfusion for 7 min with either 0.1 mmol/L NaHS (H2S donor), 10 micromol/L SNAP (NO/SNO donor), or both, followed by reperfusion with regular perfusion buffer for total 90 min. Compared to control, PostC with either NaHS or SNAP significantly reduced post-ischemic contractile dysfunction, the post-ischemic heart rate pressure product (RPP) recovery was 52.34.8% for PostC-NaHS, 51.73.9% for PostC-SNAP vs 36.42.5% for control (n=8 in each group). The post-ischemic myocardial infarction was decreased from 49.91.4% for control to 34.62.9% for PostC-NaHS and 35.22.9% for PostC-SNAP. Interestingly, PostC simultaneously with two donors together had a synergistic protective effect, post-ischemic RPP recovery was 72.24.2% and infarct size was 19.73.0%. Dilated idiopathic cardiomyopathy (DCM) is one of the most common types of cardiomyopathy. Sex differences in hypertrophy are well known. We examine the role of sex differences in S-nitrosylation and oxidation from explanted DCM and non-failing donor male and female human hearts. Redox-based resin-assisted capture for oxidation (Ox-RAC) and SNO (SNO-RAC) proteomic analysis were used to measure protein oxidation and SNO respectively. In addition, a 2D-DIGE using DyLight-maleimide sulfhydryl-reactive fuors was used to identify the SNO proteins. Protein oxidation increased in DCM biopsies in comparison with healthy donors. Interestingly we did not find a consistent decrease in SNO in failing hearts; we found that some proteins showed an increase in SNO and others a decrease and there were sex differences in the response. We found 10 proteins with a significant decrease in SNO and 4 proteins with an increase in SNO in failing female hearts. Comparing non-failing and failing male hearts we found 9 proteins with a significant decrease and 12 proteins with a significant increase. We also found an increase in S-glutathiolation of eNOS in failing female versus male hearts, suggesting an increase in uncoupled NOS in females. These findings highlight the importance of the nitroso/redox signaling in both physiological and pathological conditions, suggesting a potential target to treat HF.It has been proposed that an increase in oxidative stress in heart failure leads to a decrease in nitric oxide signaling, leading to impaired nitroso-redox signaling. To test this hypothesis we investigated the occurrence of protein S-nitrosylation (SNO) and protein oxidation in biopsies from explanted DCM and non-failing donor male and female human hearts. A modied biotin switch method using DyLight-maleimide sulfhydryl-reactive fluors was used to identify the SNO proteins and a resin-assisted capture Ox-RAC was used to measure protein oxidation. We found that oxidative stress rises in DCM human biopsies in comparison with healthy donors. DyLight-maleimide gel electrophoresis showed that females have an increase in protein SNO compared with males at baseline. Interestingly many of the proteins that showed an increase in SNO in females are mitochondrial proteins. Because there are sex differences in SNO at baseline, we examined changes in SNO in heart failure as a function of sex and found sex differences in this response.